Biomedical subjects
E Vuorio
Publications and source records attributed to E Vuorio.
Expression of type I and III collagen and laminin beta1 after rat sciatic nerve crush injury.
Extracellular matrix changes are thought to be essential to the regeneration of peripheral nerves. The production of this matrix is believed to be regulated by interactions between axons and their supporting cells. In this study matrix production and cell proliferation were studied during rat sciatic nerve regeneration after a crush injury, and compared to that after rat sciatic nerve transection. Expression of proalpha1(I) and proalpha1(III) collagen and laminin beta1 mRNAs was followed in isolated endoneuria by Northern and in situ hybridization both proximally and distally to the site of either a crush injury or transection of rat sciatic nerve up to 18 weeks. Changes in the Schwann cell and fibroblast populations were monitored by morphometric analysis of endoneurial cross-sections immunostained for S-100 protein. The process of axonal regeneration was followed by Bielschowsky's silver staining. A crush injury initially resulted in increased expression of all mRNAs studied in the endoneurial cells. However, with progressing axonal regeneration the amount of collagen mRNAs returned to control levels, whereas the amount of laminin beta1 mRNA in the distal site of the crush remained elevated throughout the study period. The expression of type I collagen mRNA was enhanced after nerve transection injury compared to that after the crush injury. The epineurial fibroblasts actively expressed both type I and III collagen mRNAs after the injury. The proliferation of Schwann cells and the expression of collagen mRNAs are not, at least directly, related to the axonal regeneration. However, the long-lasting and strong expression of laminin beta1 mRNA after a nerve crush injury may be related to good axonal regeneration. The expression of type I collagen in the epineurium may lead to clinically well-recognized epineurial scarring and thus impede axonal regeneration.
Retarded skeletal development in transgenic mice with a type II collagen mutation.
Transgenic mice harboring specific mutations provide a tool for systematic studies on the effects of dominant mutations on embryonic development and growth. In the present study, several different techniques were employed to follow the development of growth abnormalities and other phenotypic consequences in the mouse litters from matings of heterozygous Del1 mice carrying six copies of a mouse type II collagen transgene with a small engineered deletion mutation. Skeletal staining of complete litters with alcian blue/alizarin red S revealed that the onset of chondrogenesis and subsequent endochondral ossification were delayed in heterozygous and homozygous Del1 embryos. The lengths, widths, and shapes of long bones were all affected. Histological and in situ hybridization analyses revealed several types of abnormalities in the epiphyseal growth plates of homozygous Del1 embryos including disorganization of growth plate architecture, abnormal appositional growth activity, an increase in the number of hypertrophic chondrocytes, a deficiency in the formation of metaphyseal cancellous bone, and development of necrotic areas in the epiphyseal heads. Many of these findings parallel those seen in human chondrodysplasias. The basic information obtained on the effects of a specific deletion mutation in the type II collagen gene on the development and growth of the transgenic embryos provides a background for testing the efficacy of novel therapeutic strategies for prevention of such growth abnormalities.
Conservation of the sizes of 53 introns and over 100 intronic sequences for the binding of common transcription factors in the human and mouse genes for type II procollagen (COL2A1).
Over 11,000 bp of previously undefined sequences of the human COL2A1 gene were defined. The results made it possible to compare the intron structures of a highly complex gene from man and mouse. Surprisingly, the sizes of the 53 introns of the two genes were highly conserved with a mean difference of 13%. After alignment of the sequences, 69% of the intron sequences were identical. The introns contained consensus sequences for the binding of over 100 different transcription factors that were conserved in the introns of the two genes. The first intron of the gene contained 80 conserved consensus sequences and the remaining 52 introns of the gene contained 106 conserved sequences for the binding of transcription factors. The 5'-end of intron 2 in both genes had a potential for forming a stem loop in RNA transcripts.
Expression of type VI, IX and XI collagen genes and alternative splicing of type II collagen transcripts in fracture callus tissue in mice.
The levels of six mRNAs coding for constituent alpha-chains of three minor collagens of cartilage were analyzed in an experimental fracture model in normal and transgenic Del1 mice harboring a deletion mutation of exon 7 in the type II collagen gene. Reduced and retarded chondrogenesis in Del1 mice was evident in callus samples as reduced mRNA levels for the cartilage specific type IX and XI collagens at days 7 and 9 of fracture healing. Analysis of the calluses for alternative splicing of pro alpha 1(II) collagen mRNA also suggested retarded chondrogenesis in Del1 calluses. Another developmentally regulated step in limb development, a switch between alternative promoters of the alpha 1(IX) collagen gene, was also seen during fracture healing but was less obvious in Del1 calluses. Finally, the current data suggest that the abnormality in bone remodelling in Del1 mice involves activation of the genes coding for alpha 1(XI) and alpha 2(VI) collagens.
Developmental expression of a type II collagen/beta-galactosidase fusion gene in transgenic mice.
The correct temporal and spatial expression of the type II collagen gene is believed to be important for normal development and growth of the skeleton and the eye, i.e., tissues where the protein product is predominantly found. To study transcriptional activation of type II collagen gene in skeletal and nonskeletal tissues we produced transgenic mice carrying murine proalpha1(II) collagen/beta-galactosidase fusion gene constructs. The expression of the fusion gene was found to depend on the presence of intron 1 deleted failed to reveal any beta-galactosidase activity confirming the important role of regulatory sequences within intron 1 of the gene. High-level expression of the functional construct was clearly confined to cartilaginous tissues but transient low-level expression was also observed in extraskeletal locations, such as the developing brain and the notochord. The results demonstrate that the regulatory elements in the proalpha1(II) collagen/beta-galactosidase fusion gene construct confer both temporal and spatial specificity indistinguishable from that of the endogenous proalpha1(II) collagen gene as determined by the presence of the corresponding mRNA by in situ hybridization. Furthermore the beta-galactosidase activity correlated well with the progression of chondrogenesis as seen by staining of whole mouse embryos with Alizarin red S and Alcian blue in the hybrid mouse strain used for microinjections. The transgenic mouse line produced should prove useful for studies on various aspects of chondrogenesis. Furthermore, the data shows that the regulatory elements present in the construct are sufficient for targetting the expression of other genes in cartilage.
Coordinate patterns of expression of type I and III collagens during mouse development.
The extracellular proteins types I and III collagen are abundantly expressed during development. Here, the patterns of the pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen mRNAs are systematically examined from 7.5 to 17.5 days of development (E7.5 to E17.5) in the mouse using in situ hybridization with specific riboprobes. Coordinated expression of pro alpha 1(I) and pro alpha 2(I) collagen mRNA was found throughout development in all regions examined. Widespread type I collagen expression starting at E8.5 occurred in embryonic mesoderm, sclerotomes, dermatomes, and in the forming connective tissues. After E14.5, regions of ossification showed highest levels of type I collagen expression. Pro alpha 1(III) collagen expression was specific to and coordinated with patterns of type I collagen expression in many fibroblast-containing tissues. No expression of type III collagen occurred in osteoblasts. This comprehensive study of the transcripts of abundantly expressed structural proteins should provide a basis for comparison of other key extracellular matrix molecules and serve as a reference for studies on the patterns of activities of various promoter/enhancer-reporter gene constructions of type I and III collagen genes in transgenic mice.
Extended expression of cartilage components in experimental pseudoarthrosis.
The healing of femoral fractures in an experimental rat pseudoarthrosis model was followed by studying the expression of cartilage specific genes coding for type II and X collagens and aggrecan, soft tissue and bone specific type I collagen, and decorin. Severe impairment of healing was observed with cartilage gene expression continuing until the seventh week and then declining rapidly. The abnormal healing pattern results in an inactive scar-like callus after the ninth week of healing even though house-keeping (e.g., GAPDH) genes are continuously expressed in the tissue. These results could be explained on the basis of continuous chondrogenic stimulus extending much beyond the normal range. If union is not achieved because of mechanical instability, signal of endochondral ossification persists until it becomes exhausted and callus at the fracture gap becomes an inactive fibrous scar. The disturbed matrix gene expression was confirmed by histology.
Expression of matrix genes during incorporation of cancellous bone allografts and autografts.
The healing process of cylindrical metaphyseal bone defects filled with fresh autogeneic or frozen allogeneic cancellous bone grafts was studied using histology, biomechanical testing, and molecular biology. The results were compared with the healing pattern observed in nongrafted defects, where a strong initial expression of Type III collagen was followed by slowly starting expression of osteonectin and Type 1 collagen genes. Autogeneic or allogeneic cancellous bone grafts considerably increased the healing rate of the defects. The sequence of healing was similar with both types of grafts, but the maximal levels of Type III and Type I collagen messenger ribonucleic acids (mRNAs) were delayed by 1 week when allografts were used. Histologically, during the initial phase the grafts had an invasion of progenitor mesenchymal tissue within 2 weeks followed by intramembranous bone formation and by a gradual decline in Type I collagen expression. Osteonectin mRNA was present throughout the healing process, but no expression of Type II collagen was seen. Indentation testing showed no significant biomechanical differences between the autografts and allografts. Histology and molecular biology testing results revealed that the production of extracellular matrix starts slower in allogeneic cancellous bone grafts than in autografts, suggesting a basic difference in the initial host tissue response between autogeneic and allogeneic bone grafts.
Transgenic mice with deficiencies in cartilage collagens: possible models for gene therapy.
We address three issues that are important when considering somatic gene therapy approaches to osteoarthritis (OA) and related syndromes. First, only those diseases for which a precise molecular etiology has been established should be contemplated for somatic gene therapy. Second, DNA sequences should be identified that restrict expression of correcting genes to chondrocytes; we discuss the use of transgenic mice to identify such sequences. Third, we emphasize the usefulness of establishing animal models that mimic human OA syndromes by genetic manipulations. These transgenic models should be essential for testing gene therapy approaches in vivo.
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Analysis of aggrecan and tenascin gene expression in mouse skeletal tissues by northern and in situ hybridization using species specific cDNA probes.
Cartilage matrix is an interacting multicomponent system of collagen fibrils, fibril-associated small proteoglycans, and large proteoglycans and glycoproteins entrapped within the fibrillar network. In order to better understand the relationships between these different components we have constructed short cDNA clones for detection of mRNAs for two major noncollagenous macromolecules of cartilage matrix, aggrecan and tenascin. We subsequently determined their corresponding mRNA levels by Northern analysis in a panel of total RNAs isolated from several newborn mouse tissues. The expression of aggrecan was strictly restricted to cartilages while tenascin mRNA was present at variable levels in most of the tissues studied. The cDNA clones were also used to identify the cells responsible for aggrecan and tenascin production in newborn mouse tissues by in situ hybridization. With this technique aggrecan mRNA was detected in chondrocytes throughout the developing skeleton in a pattern very similar but not identical to those of type II and IX collagen mRNAs. In the newborn mouse skeleton tenascin and aggrecan mRNAs were expressed essentially in a mutually exclusive manner, tenascin transcripts being present in osteoblasts, periosteal and perichondrial cells, and in cells at articular surfaces. None of these cells expressed the cartilage specific collagen or aggrecan genes. The results further suggest different patterns of gene expression in chondrocytes based on their location in the different cartilages.
The genes encoding alpha 2(IX) collagen (COL9A2) map to human chromosome 1p32.3-p33 and mouse chromosome 4.
We have determined the chromosomal locations of the human and murine genes coding for alpha 2(IX) collagen, a polypeptide subunit of the heterotrimeric type IX collagen molecule. COL9A2 was mapped to human chromosome 1p32.3-p33 using fluorescence in situ hybridization. A single-strand conformational polymorphism within the murine Col9a2 gene was used to map this locus to mouse chromosome 4. We also present new sequence data, which completes the coding information for the human alpha 2(IX) chain and revises the sequence for the chicken alpha 2(IX) chain. This permits comparison of the carboxyl-terminal (NC1) domains of the alpha 1(IX), alpha 2(IX), and alpha 3(IX) chains across several species.
The exon structure of the mouse alpha 2(IX) collagen gene shows unexpected divergence from the chick gene.
One cosmid and two overlapping phage clones covering the entire mouse alpha 2(IX) collagen gene including 12 kilobase pairs (kb) of 5'- and 8 kb of 3'-flanking sequences were isolated from two genomic libraries. The overall gene structure was determined by restriction mapping and nucleotide sequencing. The gene spans 16 kb from the start of transcription to the polyadenylation site and contains 32 exons. It codes for a mRNA of 3 kb that translates into a polypeptide of 688 amino acids. The intron-exon junctions and mRNA structure were confirmed by amplification of cDNA made for mouse cartilage RNA. The coding sequence of the mouse alpha 2(IX) collagen gene shows marked similarities to those for other type IX collagen chains. Although the overall exon-intron organization of the mouse gene is very similar to the chick alpha 2(IX) gene, some unexpected differences were observed at the splice junctions. Split codons characteristic for the central triple helical domain of the chick were not found in the mouse gene that thus exhibited a long stretch of exons with sizes that are multiples of 9 base pairs in this domain. The promoter of the mouse alpha 2(IX) collagen gene contains some G + C-rich elements including three Sp1 consensus recognition sites and a far upstream CCAAT box but no TATAA box. Both primer extension and RNase protection assays revealed several transcription start sites within 418 base pairs of the promoter. The present study reports the first complete nucleotide sequence of any type IX collagen gene and forms the basis for comparative structural studies on this collagen type and for experiments involving transgenic mice.
Cloning of cDNA for rat pro alpha 1(III) collagen mRNA. Different expression patterns of type I and type III collagen and fibronectin genes in experimental granulation tissue.
A cDNA clone for rat pro alpha 1(III) collagen mRNA was isolated from a cDNA library constructed for poly(A)+ RNA from 15-day experimental granulation tissue. Two clones, pRGR1 and pRGR5, were characterized by restriction mapping and sequencing. Comparison with human type III collagen sequences revealed 92% identity at the level of translated amino acids, and 88% identity at nucleotide level in the coding region. In the 3'-untranslated sequence the identity was even higher (90%). The clones were used together with cDNA clones for type I collagen chains, fibronectin and gamma-actin to study the expression of the corresponding mRNAs during the development of experimental sponge-induced granulation tissue in rats. These studies revealed a marked activation of type I and type III collagen genes during the second week of granuloma development followed by a transient reduction in their levels during the third week. The mRNA levels for both collagen types remained relatively unchanged from day 25. The molar ratio of pro alpha 1(III) and pro alpha 1(I) collagen mRNAs was at a maximum on day 6, and then decreased to reach a plateau by the end of the third week. Fibronectin mRNA levels were found to increase slower; the maximum value was reached during the fifth week of granuloma development. The mRNA levels of gamma-actin increased continuously up to the end of the fourth week, thus following the cellular maturation of the tissue.
Retarded chondrogenesis in transgenic mice with a type II collagen defect results in fracture healing abnormalities.
We have examined the biological and biomechanical consequences of defective type II collagen production for fracture repair employing a genetically engineered mouse line Del1 which was generated by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7 (Metsäranta et al. [1992] J. Cell Biol. 118:203-212). Standardized tibial fractures were produced in transgenic Del1 mice and their nontransgenic littermates were used as controls. The fracture callus tissues were analyzed at days 7, 9, 14, 28, and 42 using radiography, histomorphometry, biomechanical testing, and Northern analysis of mRNAs for several tissue-specific matrix components. Deficient production of cartilage in Del1 mice resulted in reduced radiographic callus size, smaller cross-sectional area, and impaired biomechanical properties when compared with fractures of nontransgenic control mice. The differences were most evident in 14-day fracture calluses. Consequently mRNAs for cartilage-specific type IX and X collagens and aggrecan were also reduced in Del1 calluses. Levels of type II collagen mRNAs were unaffected since the mutated transgene produced additional type II collagen mRNA molecules. Further abnormalities in the fracture repair process of Del1 mice were observed in callus remodeling. In the control animals a typical feature of external callus remodeling was reduction of callus size during endochondral ossification between days 14 and 28. Such reduction was not observed in the transgenic mice. Histological examination of fracture calluses suggested also a reduction in trabecular surface area, which was found to be even more pronounced in metaphyseal bone of Del1 mice. Despite these differences the biomechanical properties of the calluses in the two groups became similar by day 28 of fracture healing. The results thus suggest that reduced chondrogenesis due to the presence of mutated transgenes in Del1 mice not only causes a temporary impairment in biomechanical properties of healing fractures but also affects later stages of callus remodeling.
Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice.
Studies on the function of extracellular matrix components of cartilages and on chondrocyte-specific regulatory mechanisms will benefit from approaches in which transgenic mice and cell cultures will complement each other. We therefore established and extensively characterized primary cultures of mouse chondrocytes isolated from rib growth plates of newborn mice harboring a transgene in which type II collagen gene regulatory sequences were driving expression of an E. coli beta-galactosidase reporter gene. Primary chondrocytes expressed a fully differentiated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synthesized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondrocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they displayed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with that of cartilage-specific genes, and started expressing high levels of type I collagen RNA. The use of transgenic chondrocytes allowed us to easily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as those reported here, should also help characterize biochemically the phenotypes of other transgenic mice in studies of genetic diseases of cartilages and of mechanisms involved in chondrogenesis.
Differential expression of myc, max and RB1 genes in human gliomas and glioma cell lines.
Deregulated expression of myc proto-oncogenes is implicated in several human neoplasias. We analysed the expression of c-myc, N-myc, L-myc, max and RB1 mRNAs in a panel of human gliomas and glioma cell lines and compared the findings with normal neural cells. The max and RB1 genes were included in the study because their protein products can interact with the Myc proteins, being thus putative modulators of Myc activity. Several gliomas contained c/L-myc mRNAs at levels higher than those in fetal brain, L-myc predominantly in grade II/III and c-myc in grade III gliomas. High-level N-myc expression was detected. In one small-cell glioblastoma and lower levels in five other gliomas. In contrast, glioma cell lines totally lacked N/L-myc expression. The in situ hybridisations revealed mutually exclusive topographic distribution of myc and glial fibrillary acidic protein (GFAP) mRNAs, and a lack of correlation between myc expression and proliferative activity, max and RB1 mRNAs were detected in most tumours and cell lines. The glioma cells displayed interesting alternative splicing patterns of max mRNAs encoding Max proteins which either suppress (Max) or augment (delta Max) the transforming activity of Myc. We conclude that (1) glioma cells in vivo may coexpress several myc genes, thus resembling fetal neural cells; but (2) cultured glioma cells expression only c-myc; (3) myc, max and RB1 are regulated independently in glioma cells; and (4) alternative processing of max mRNA in some glioma cells results in delta Max encoding mRNAs not seen in normal fetal brain.